The use of nanopores, including biological nanopores, for detection of single molecules has been in practice for two decades (Deamer, D. W. Branton, D., “Characterization of Nucleic Acids by Nanopore Analysis,” Acc. Chem. Res. 2002, 35, 817-825). The biological protein nanopore α-hemolysin (αHL) from Staphylococcus aureus has proven to be ideal for single molecule detection, given the inner pore constriction diameter of ˜1.4 nm (Song, S., Hobaugh, M. R., Shustak, C., Cheley, S., Bayley, H., Govaux, J. E., “Structure of Staphylococcal α-Hemolysin, a Hepatmeric Transmembrane Pore,” Science, 1996, 274, 1859-1865).
By imbedding αHL into a lipid bilayer, the ionic resistance through the ion channel can be measured. αHL can be chemically modified or genetically engineered to selectively bind analyte molecules. Fluctuations in the resistance across a single channel can also be monitored as single molecules bind to the protein. These fluctuations are molecule specific allowing for the simultaneous detection and characterization of multiple analytes (Bayley, H, Cremer, P. S. “Stochastic Sensors Inspired by Biology,” Nature, 2001, 413, 226-230)
Recently, the use of biological nanopores such as αHL for the detection and sequencing of DNA has been investigated (Kasianowicz, J. J., Brandin, E., Branton, D., Deamer, D. W., “Characterization of Individual Polynucleotide Molecules Using a Membrane Channel,” Proc. Natl. Acad. Sci. 1996, 93, 13770-13773). Simple proof of concept experiments, where the number of nucleotides in the single stranded DNA (ssDNA) was identified by measuring the length of time the strand spends in the ion channel, were conducted. By applying a small voltage, Deamer and coworkers were able to drive single DNA molecules through an ion channel that was inserted in a lipid bilayer. As the molecule translocates through the channel, a transient decrease in current is observed due to the DNA blocking the motion of the charge-carrying electrolyte ions.
Protein ion channel investigations rely on the formation of a lipid bilayer across a 15 μm to 150 μm diameter orifice in a thin TEFLON® or other polymer-based membrane. There are several drawbacks associated with this single-ion measurement platform due to the large area of the orifice. First, the lipid bilayer spanning the orifice of the Teflon or other polymer-based membrane is very susceptible to failure due to vibrations, pressure change and voltage fluctuations. Second, measurements in conventional bilayer systems indicate that the bilayer is not stable but in a state of continuous thinning. Even if extensive precautions are taken to minimize vibration and voltage disturbances, the bilayer lifetime is typically only a few hours. These robustness and lifetime problems are generally recognized as the key current roadblocks in developing usable sensors or sequencing devices based on protein ion channels, rule out and any type of portable or moveable system.
A more robust sensor platform capable of supporting the bilayer structure would expose a sub-microscopic bilayer region (e.g., 1 μm2 or smaller area) for the insertion of a protein channel. The small area would reduce failures due to mechanical and electrical disturbances, and has been shown to allow lifetimes of greater than 20 days in preliminary tests. Such a device would allow for less complicated apparatus that does not require bulky and expensive vibration isolation, much longer duration operation, and potentially portable systems for applications of ion channels in drug development, biosensing, DNA sequencing, etc.